Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar.

BACKGROUND
A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.


METHODS
A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.


RESULTS
With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.


CONCLUSION
The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.


Introduction
number of methods for detecting the diversity in Entamoeba have been described over the years (1), but recently Ali et al. investigated the association between the genotypes of parasites and the clinical outcome of infection (2) using a 6-locus genotyping system based on tRNA-linked short tandem repeats (STRs) (3). The six targets for amplification in this method (Loci S-Q, D-A, A-L, S TGA -D, R-R and N-K2) were selected from among over 40 STR-containing loci linked to tRNA genes in E. histolytica (1). One of these polymorphic loci is Locus D-A, previously called locus 1-2, has been shown to be potentially useful for investigating the molecular epidemiology of E. histolytica and E. dispar (4,5). Zaki et al. isolated and characterized locus D-A, and later compared the nucleotide sequence of this locus between E. dispar and E. histolytica. This revealed significant differences in both the STRs and the flanking regions (6,7). Haghighi et al. analyzed the genetic polymorphisms of four loci, including D-A, 5-6 among 79 isolates of E. histolytica obtained from different geographic regions. They reported large scale genetic differentiation between Japanese and Thai isolates (4,8). Recently, molecular studies have been extended to distinguish and investigate the distribution of these two species in Iran (9)(10)(11). In the present study, genetic polymorphism of another tRNA-liked STR-containing locus, A-L, was analyzed using PCR and sequencing methods in order to clarify further the genotypic differences among E. dispar isolates.

Materials and Methods
A total of 28 E. dispar strains were analyzed. Twenty four of them were collected from Iranian patients referred to the clinical laboratories of hospitals in the city of Tehran and Zahedan and 4 strains were collected from asymptomatic individuals referred to health care centers in the city of Gonbad (5). Clinical information on the samples is given in Table 1. All the samples used in this study were diagnosed as positive for Entamoeba spp. by microscopic examination of fresh stools using direct smears, formalinether concentrated, and trichrome stained specimens. The genomic DNA was extracted directly from stool and samples were identified to species level by locus D-A based PCR analysis, as previously described (5). For genotype analysis, Locus A-L was amplified by PCR with the primer set 5′-CATCTCCAT TATTATGTATCTATTTATC-TATTTA-3′and 5′-GGCACGAATGCTTTGA-TATATAA-3′ (3). PCR products were analyzed by electrophoresis using 1.8% agarose gels (Fermentas, #R0491) in Tris-boric acid-EDTA buffer containing ethidium bromide after which the gels were photographed under ultraviolet light (UVIdoc, UVItec Limited, Cambridge, United Kingdom). The PCR products were sequenced using the amplification primers and an Applied Biosystems (ABI) Terminator Cycle Sequencing Ready Reaction kit (BigDye® Terminator V3.1 Cycle Sequencing Kit) on an ABI 3130xl Genetic Analyzer. The sequences obtained were edited manually and aligned using Gene Runner software (version 3.05). Nucleotide sequences, except forward and reverse primer regions, were aligned with the only previously available locus A-L sequence from E. dispar in GenBank (AY842969). All sequences were submitted to the GenBank/EMBL/DDBJ database under accession numbers HQ439931-HQ439958.

Results
In order to investigate genetic diversity, the PCR-amplified products from 28 E. dispar isolates were subjected to direct sequencing. Samples were sequenced in both directions and, when any variations were found, results were confirmed by sequencing of at least two independent PCR products. All sequences were analyzed by Chromas version 1.45 (Technelysium, Queensland, Australia) and the sequence homology was compared with the sequences in GenBank by BLAST analysis. PCR amplification and sequencing of the PCR products showed a remarkable level of genetic diversity in size, number and position of the repeat units among the E. dispar isolates (Fig. 1). Nucleotide sequence length varied from 448bp to 507bp which led to visible differences in PCR product size (Table 1). Seven distinct nucleotide sequences were obtained from the isolates while gel analysis of the PCR products show three groups distinguishable by size. Sequence E represents the dominant genotype (11/28, 37%) among the Iranian isolates and its 472 bp fragment was also the most frequent size found. The STR organization in locus A-L from E. dispar SAW760 (AY842969), which has 507 nucleotides, is compared to the organization in sequences from the Iranian isolates in Fig. 1.

Discussion
The ability to identify strains of Entamoeba dispar may lead to insights into the population structure and epidemiology of the organism. When polymorphism in two E. histolytica loci, 1-2 and 5-6, was studied in 2001 by Zaki et al., the remarkable diversity in length, type and numbers of the repeat units found showed that they have the potential to allow the investigation of genetic differences between invasive and noninvasive E. histolytica isolates. Sequences corresponding to the polymorphic loci reported from E. histolytica have also been detected in E. dispar. Comparison of nucleotide sequences in two loci between E. dispar and E. histolytica revealed significant differences in both the repeats and the flanking regions, which allowed the typing and differentiation of these two parasites simultaneously (6,7). However although variation in locus A-L has been investigated to some extent in E. histolytica (12), it has not been investigated previously in E. dispar. The tRNA gene regions in locus A-L are conserved and are the site of the primers used, but in the middle there are repeat units of between 8 and 15 nucleotides which vary among isolates. Elimination, duplication and substitution of units in this repeat-containing region are the basis of polymorphisms detected in the two species. In our previous study PCR amplification of locus D-A among Iranian E. dispar isolates, showed a remarkable genetic diversity in size and this result confirmed by Sequencing of PCR products (5). By simultaneous investigation of locus A-L and locus D-A (5), 26 subtypes out of 28 Entamoeba dispar isolates were distinguished (the molecular patterns of NH19IR and NH20IR, also NHM2IR and NHM3IR are not different in two loci) ( Table 1).
In this study, no meaningful correlation between infection with E. dispar and age, sex or parasite genotype was observed. However, it appears that sequence type E is overrepresented in the male individuals compared to females (out of 11 individuals who showed this type, 8 of these were males) or sequence type E is common in asymptomatic patients. In 2001, in Bangladesh, the role of genetic diversity in E. histolytica virulence was studied and it was clarified that the genetic diversity of E. histolytica subspecies in endemic regions is because of SREHP polymorphism. Noticeably, the polymorphism of liver amebiasis subspecies was different from intestinal amebiasis subspecies (13)(14). Haghighi et al. reported a considerable polymorphic in size, number and position of the repeat units in four loci (1-2, 5-6, SREHP and Chitinase) of different E. histolytica isolates obtained from stool samples of mentally handicapped individuals and male homosexuals from different regions of Japan (4,8). They proposed that genotyping of ameba isolates should help to determine geographic origins of isolates and routes of transmission. Although the studies of Haghighi et al. did not detect a link between genotype and symptoms, their samples were from geographically diverse sources and acquired over a number of years (4,8) . The studies of Ali et al. developed a reliable method for PCR-based genotyping of E. histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species and suggesting that the parasite genome does contribute in some way to the outcome of infection with E. histolytica (2,3). In conclusion, we propose that molecular typing and analysis of genotypes of E. histolytica and E. dispar isolates from a variety of locations should help in determining the geographic origins of isolates and routes of transmission.